We isolated total exosomes (30–100 nm fin diameter) from serum samples obtained from ME/CFS patients and controls as we reported previously. 28 Interestingly, we reported less total EV-associated protein obtained from serum of ME/CFS patients than controls unlike two other studies that reported no significant difference in EV yields obtained from plasma of ME/CFS patients and controls. 29, 30 In contrast, two other studies reported a significant increase in EV content in the serum of ME/CFS patients and controls. 31, 32 These apparent differences may depend on the activity status of the controls, use of serum or plasma, as well as method of EV isolation and purification, thus highlighting the need for standardization for future studies.
Exosomes are EVs with a size range of 30–100 nm in diameter and may carry different cargo than larger EVs (100–1000 nm in diameter). 24 EVs have been implicated in brain disorders,26,27 but their role in ME/CFS had not been adequately investigated. EVs have been isolated from ME/CFS patients and characterized especially for their content of microRNA. 30 Another study investigated the cytokine profile of plasma and MEs isolated from plasma of ME/CFS patients and reported no significant differences. 29 Hence, the amount of circulating cytokines whether free or EV=-associated may not be important, as compared to the ability of EVs to potentially stimulate the release of pro-inflammatory cytokines from microglia in the brain.
The source of exosomes in ME/CFS patients in not presently known. One possibility is that they derive from mast cells 33,34 either in the hypothalamus where they are particularly abundant 35, 36 perivascularly in close proximity to neurons 36, 37 or from peripheral mast cells and enter the brain by crossing the blood-brain barrier (BBB), which is known to be disrupted in neuropsychiatric disorders. 38,39 Mast cells and their mediators have been implicated in diseases comorbid with ME/CFS. 40 Moreover, mast cells are increased in the skin of patients with ME/CFS, 41, 42 who also show increased skin hypersensitivity. 43 Hyper-responsiveness of the bronchi, implying activation of mast cells, has also been noted in ME/CFS patients. 44 Activated mast cells could release additional mediators contributing to ME/CFS symptoms, 45, 46 or mtDNA. 47 The possible mast cell source of the exosomes could be determined in the future by Western blot analysis of the presence in exosomes of the mast cell-specific surface markers (FceRI, MRGX2 and CD-117). 34
Here we show that serum mitochondrial DNA (mtDNA), associated with exosomes, is increased after exercise in patients with ME/CFS as compared to unrelated, healthy controls. Moreover, we report that exosomes containing mtDNA from patients with ME/CFS stimulate cultured human microglia to secrete IL-1b. We had reported that exosomes purified from serum from children with autism spectrum disorder (ASD) contained mtDNA and stimulated human microglia to secrete IL-1b.28 This key pro-inflammatory cytokine has been implicated in fatigue 48 and in a rat model of CFS. 49 We had also reported that extracellular mtDNA is increased in the serum of children with ASD.50
Mitochondrial DNA could act as an “innate pathogen” leading to a localized auto-inflammatory response in the hypothalamus.51–54 Analysis of the mitochondrial genome in ME/CFS cases indicated that individuals with a certain haplotype were more likely to exhibit certain neurologic symptoms, but there was no association with either susceptibility 55 or disease severity. 56 An unusual pattern of mtDNA deletions was reported in the skeletal muscle of one patient with ME/CFS. 57 Cellular bioenergetics has been reported to be impaired in patients with ME/CFS 58 and salivary mtDNA was found to be decreased in subjects with fatigue. 59 Even though mtDNA was shown to be neurotoxic in rat brain slices, 60–62 the mtDNA molecular patterns N-formyl peptides and cardiolipin did not stimulate IL-6 or TNF release from HMC-3 microglia 63 implying that the entire mtDNA may be required for stimulation.
There are a number of limitations in this study. First, the number of subjects analyzed was small. Pathogenetic changes in ME/CFS patients may only occur in the brain, especially the hypothalamus, in which case we will have to await the availability of brain tissue to carry out analysis of the proposed biomarkers, as we recently reported for autism spectrum disorder. 64